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Characterization of NAD-dependent malate dehydrogenases from spinach leaves

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Summary.

Spinach leaves were used to extract isoforms of NAD-dependent malate dehydrogenase (NAD-MDH) (EC 1.1.1.37), either soluble or bound to microsomal, plasma, or chloroplast envelope membranes. All fractions were subjected to isoelectric focusing analysis, which showed that purified chloroplast envelopes contain an NAD-MDH isoform tightly bound to the membranes, since treatment with 0.5 or 1% Triton X-100 was not able to release the enzyme from the envelopes. In contrast, plasma membranes released an isoform with a pI of 3.5 following treatment with 0.5% Triton X-100. The most abundant soluble leaf isoform had a pI of 9, while the chloroplast stroma contained an isoform with a pI of 5.3. Kinetic analysis of oxaloacetate (OAA)-dependent NADH oxidation in different fractions gave different K m values for both substrates, the envelope- and plasma membrane-bound NAD-MDH exhibiting the highest affinities for OAA. Leaf plasma membrane-bound MDH exhibited a high capacity for both reaction directions (malate oxidation and OAA reduction), while the two chloroplast isoforms (stromal and envelope-bound) preferentially reduced OAA. Our results indicate that the chloroplast envelope contains a specifically attached NAD-MDH isoform that could provide direct coupling between chloroplast and cytosol adenylate pools.

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Abbreviations

MDH:

malate dehydrogenase

NAD-MDH:

NAD-dependent malate dehydrogenase

HPR:

hydroxypyruvate reductase

IEF:

isoelectric focusing

OAA:

oxaloacetate

ADH:

alcohol dehydrogenase

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Authors and Affiliations

  1. Faculty of Biology, University of Belgrade, Belgrade

    T. Cvetić

  2. Center for Multidisciplinary Studies, University of Belgrade, Belgrade

    S. Veljović-Jovanović & Ž. Vučinić

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  1. T. Cvetić
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  2. S. Veljović-Jovanović
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  3. Ž. Vučinić
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Correspondence to T. Cvetić.

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Correspondence: T. Cvetić, Institute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia.

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Cvetić, T., Veljović-Jovanović, S. & Vučinić, Ž. Characterization of NAD-dependent malate dehydrogenases from spinach leaves. Protoplasma 232, 247–253 (2008). https://doi.org/10.1007/s00709-007-0282-7

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  • DOI: https://doi.org/10.1007/s00709-007-0282-7

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